CALCIUM-DEPENDENT potassium (K-Ca) channels carry ionic currents that regulate important cellular functions(1-3). Like some other ion channels(4-10), K-Ca channels are modulated by protein phosphorylation(2,11,15-21). The recent cloning of complementary DNAs encoding Slo K-Ca channels(12-14) has enabled K-Ca channel modulation to be investigated. We report here that protein phosphorylation modulates the activity of Drosophila Slo K-Ca channels expressed in Xenopus oocytes. Application of ATP-gamma S to detached membrane patches increases Slo channel activity by shifting channel voltage sensitivity. This modulation is blocked by a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Mutation of a single serine residue in the channel protein also blocks modulation by ATP-gamma S, demonstrating that phosphorylation of the Slo channel protein itself modulates channel activity. The results also indicate that K-Ca channels in oocyte membrane patches can be modulated by an endogenous PKA-like protein kinase which remains functionally associated with the channels in the detached patch.